Natural habitat connectivity and organic management modulate pest dispersal, gene flow, and natural enemy communities.

The simplification and fragmentation of agricultural landscapes generate effects on insects at multiple spatial scales. As each functional group perceives and uses the habitat differently, the response of pest insects and their associated natural enemies to environmental changes varies. Therefore, landscape structure may have consequences on gene flow among pest populations in space. This study aimed to evaluate the effects of local and landscape factors, at multiple scales, on the local infestation, gene flow and broad dispersion dynamics of the pest insect Bemisia tabaci (Genn.) Middle East-Asia Minor 1 (MEAM-1, former biotype B) (Hemiptera: Aleyrodidae) and its associated natural enemies in a tropical agroecosystem. We evaluated the abundance of B. tabaci populations and their natural enemy community in 20 tomato farms in Brazil and the gene flow between farms from 2019 to 2021. Landscapes dominated by agriculture resulted in larger B. tabaci populations and higher gene flow, especially in conventional farms. A higher density of native vegetation patches disfavored pest populations, regardless of the management system. The results revealed that whitefly responds to intermediate spatial scales and that landscape factors interact with management systems to modulate whitefly populations on focal farms. Conversely, whitefly natural enemies benefited from higher amounts of natural vegetation at small spatial scales, while the connectivity between natural habitat patches was beneficial for natural enemies regardless of the distance from the focal farm. The resulting dispersion model predicts that the movement of whiteflies between farms increases as the amount of natural vegetation decreases. Our findings demonstrate that landscape features, notably landscape configuration, can mediate infestation episodes, as they affect pest insects and natural enemies in opposite ways. We also showed that landscape features interact with farm traits, which highlights the need for management strategies at multiple spatial scales. In conclusion, we demonstrated the importance of the conservation of natural areas as a key strategy for area-wide ecological pest management and the relevance of organic farming to benefit natural enemy communities in tropical agroecosystems.

How To Be a Successful Monopartite Begomovirus in a Bipartite-Dominated World: Emergence and Spread of Tomato Mottle Leaf Curl Virus in Brazil.

Begomoviruses are members of the family Geminiviridae, a large and diverse group of plant viruses characterized by a small circular single-stranded DNA genome encapsidated in twinned quasi-icosahedral virions. Cultivated tomato (Solanum lycopersicum L.) is particularly susceptible and is infected by >100 bipartite and monopartite begomoviruses worldwide. In Brazil, 25 tomato-infecting begomoviruses have been described, most of which are bipartite. Tomato mottle leaf curl virus (ToMoLCV) is one of the most important of these and was first described in the late 1990s but has not been fully characterized. Here, we show that ToMoLCV is a monopartite begomovirus with a genomic DNA similar in size and genome organization to those of DNA-A components of New World (NW) begomoviruses. Tomato plants agroinoculated with the cloned ToMoLCV genomic DNA developed typical tomato mottle leaf curl disease symptoms, thereby fulfilling Koch's postulates and confirming the monopartite nature of the ToMoLCV genome. We further show that ToMoLCV is transmitted by whiteflies, but not mechanically. Phylogenetic analyses placed ToMoLCV in a distinct and strongly supported clade with other begomoviruses from northeastern Brazil, designated the ToMoLCV lineage. Genetic analyses of the complete sequences of 87 ToMoLCV isolates revealed substantial genetic diversity, including five strain groups and seven subpopulations, consistent with a long evolutionary history. Phylogeographic models generated with partial or complete sequences predicted that the ToMoLCV emerged in northeastern Brazil >700 years ago, diversifying locally and then spreading widely in the country. Thus, ToMoLCV emerged well before the introduction of MEAM1 whiteflies, suggesting that the evolution of NW monopartite begomoviruses was facilitated by local whitefly populations and the highly susceptible tomato host. IMPORTANCE Worldwide, diseases of tomato caused by whitefly-transmitted geminiviruses (begomoviruses) cause substantial economic losses and a reliance on insecticides for management. Here, we describe the molecular and biological properties of tomato mottle leaf curl virus (ToMoLCV) from Brazil and establish that it is a NW monopartite begomovirus indigenous to northeastern Brazil. This answered a long-standing question regarding the genome of this virus, and it is part of an emerging group of these viruses in Latin America. This appears to be driven by widespread planting of the highly susceptible tomato and by local and exotic whiteflies. Our extensive phylogenetic studies placed ToMoLCV in a distinct strongly supported clade with other begomoviruses from northeastern Brazil and revealed new insights into the origin of Brazilian begomoviruses. The novel phylogeographic analysis indicated that ToMoLCV has had a long evolutionary history, emerging in northeastern Brazil >700 years ago. Finally, the tools used here (agroinoculation system and ToMoLCV-specific PCR test) and information on the biology of the virus (host range and whitefly transmission) will be useful in developing and implementing integrated pest management (IPM) programs targeting ToMoLCV.

Sweetpotato Genotypes 'CIP BRS Nuti' and 'Canadense' Are Resistant to Meloidogyne incognita, M. javanica, and M. enterolobii.

Sweetpotato is a staple crop in Brazil presenting a smaller number of pathogens and diseases in comparison with other root and tuberous crops. Root-knot nematodes are among the most serious sweetpotato root pathogens. The impact of these pathogens also extends to succeeding crops. Because in Brazil, it is common to cultivate more than one crop per season, this problem is rapidly disseminated. The aim of this study was to assess the resistance of two sweetpotato clones and four commercial sweetpotato genotypes to three different Meloidogyne species. Assays were performed under greenhouse and field conditions. 'CIP BRS Nuti' and 'Canadense' were resistant to Meloidogyne incognita, M. javanica, and M. enterolobii under greenhouse conditions. This finding was obtained based on the reproduction factor < 0.2, meaning that the initial population of 5,000 nematodes was reduced to <1,000 individuals after 90 days of inoculation. Tomato and sweetpotato cultivar Beauregard, known to be susceptible to the three nematode species, were highly damaged by the pathogens. Similar results were also observed under field conditions. To our knowledge, 'CIP BRS Nuti' and 'Canadense' are the first South American commercial cultivars with triple nematode resistance.

Nanopore sequencing of tomato mottle leaf distortion virus, a new bipartite begomovirus infecting tomato in Brazil.

During a survey in a tomato field in Luziânia (Goiás State, Brazil), a single plant with mottling, chlorotic spots, and leaf distortion was found. A new bipartite begomovirus sequence was identified using nanopore sequence technology and confirmed by Sanger sequencing. The highest nucleotide sequence identity match of the DNA-A component (2596 bases) was 81.64% with tomato golden leaf deformation virus (HM357456). Due to the current species demarcation criterion of 91% nucleotide sequence identity for DNA-A, we propose this virus to be a new member of the genus Begomovirus, named "tomato mottle leaf distortion virus".

siRNA biogenesis and advances in topically applied dsRNA for controlling virus infections in tomato plants.

A non-transgenic approach based on RNA interference was employed to induce protection against tomato mosaic virus (ToMV) infection in tomato plants. dsRNA molecules targeting the cp gene of ToMV were topically applied on plants prior to virus inoculation. Protection was dose-dependent and sequence-specific. While no protection was achieved when 0-16 µg dsRNA were used, maximum rates of resistance (60 and 63%) were observed in doses of 200 and 400 µg/plant, respectively. Similar rates were also obtained against potato virus Y when targeting its cp gene. The protection was quickly activated upon dsRNA application and lasted for up to 4 days. In contrast, no detectable antiviral response was triggered by the dsRNA from a begomovirus genome, suggesting the method is not effective against phloem-limited DNA viruses. Deep sequencing was performed to analyze the biogenesis of siRNA populations. Although long-dsRNA remained in the treated leaves for at least 10 days, its systemic movement was not observed. Conversely, dsRNA-derived siRNA populations (mainly 21- and 22-nt) were detected in non-treated leaves, which indicates endogenous processing and transport through the plant. Altogether, this study provides critical information for the development of novel tools against plant viruses; strengths and limitations inherent to the systems are discussed.

New features on the genomic organization of a novel dicistrovirus identified from the sweet potato whitefly Bemisia tabaci.

The whitefly Bemisia tabaci is an agricultural pest causing large economic losses worldwide. We analysed the genomic sequence of a new viral member of the family Dicistroviridae identified by high-throughput sequencing of total RNA extracted from whiteflies. The virus, tentatively named Bemisia-associated dicistrovirus 2 (BaDV-2), has a genome of 8012 nucleotides with a polyadenylated 3' end. In contrast to typical dicistroviruses, BaDV-2 has a genome containing three open reading frames (ORFs) encoding predicted proteins of 1078 (ORF1a), 481 (ORF1b) and 834 (ORF2) amino acids, which correspond to replicase A (containing helicase and cysteine protease domains), replicase B (a domain of an RNA-dependent RNA polymerase - RdRP) and capsid proteins, respectively. The 3' end of ORF1a contains a potential frameshift signal, suggesting that ORF1a and ORF1b may be expressed as a single polyprotein (replicaseFS), corresponding to other dicistroviruses. The BaDV-2 genomic sequence shares the highest nucleotide identity (61.1 %) with Bemisia-associated dicistrovirus 1 (BaDV-1), another dicistrovirus identified from whiteflies. The full BaDV-2 replicaseFS polyprotein clustered with aparaviruses, whereas the capsid polyprotein clustered with cripaviruses in phylogenetic analyses, as with BaDV-1. The intergenic region (IGR) between ORF1b and ORF2 is predicted to adopt a secondary structure with atypical features that resembles the dicistrovirus IGR IRES structure. Our analyses indicate that BaDV-2 is a novel dicistrovirus and that BaDV-2 together with BaDV-1 may not be appropriately grouped in any of the three currently accepted dicistrovirus genera.

Complete genome sequence of a new bipartite begomovirus infecting tomato in Brazil.

A novel bipartite begomovirus infecting begomovirus-resistant tomato plants was detected via Illumina sequencing analysis, and its genome sequence was confirmed by Sanger sequencing. The DNA-A (2627 nt) and DNA-B (2587 nt) have a genome organization that is typical of New World bipartite begomoviruses, sharing 82.5% identity with tomato golden leaf distortion virus and 75.1% identity with sida chlorotic vein virus. Based on the current classification criteria for begomoviruses, this isolate should be considered a member of a new species, and the name "tomato interveinal chlorosis virus-2" (ToICV2) is proposed for this virus.

Complete genome sequence and phylogenetic analysis of a novel dicistrovirus associated with the whitefly Bemisia tabaci.

A novel single-stranded RNA virus was detected in a whitefly (Bemisia tabaci) sample subjected to high-throughput sequencing. The 8293 nt-long genome presents a polyadenylated 3' end, and contains two ORFs encoding putative 1596 and 849 aa-long proteins. These putative proteins display significant similarity to replicase and capsid polyproteins, respectively, of discitroviruses. Its complete genome sequence shared the highest nucleotide identity (59.8%) with cricket paralysis virus (family Dicistroviridae, genus Cripavirus). Phylogenetic analyses showed that this new virus putative protein sequences clustered with those from members of Dicistroviridae. However, the replicase and capsid polyprotein sequences clustered with those of members of different genera, respectively to Aparavirus and Cripavirus. RT-PCR using newly collected adult and nymph whitefly samples confirmed the presence of this virus in field populations of B. tabaci. Genome sequence and organization, and polyproteins comparison indicate that this virus is a new species of the family Dicistroviridae. The name Bemisia-associated dicistrovirus 1 is proposed for this virus.

Erratum to: Discovery of two small circular ssDNA viruses associated with the whitefly Bemisia tabaci.

The complete genome sequences of two novel small circular DNA viruses isolated from sweet-potato whiteflies collected in Central-West (AdDF) and Southeast (AdO) regions of Brazil were determined by Next Generation Sequencing (NGS), and confirmed by cloning and Sanger sequencing. The genomes are 2,199 and 2,211 nt-long, respectively, encoding a putative coat protein (CP) and a replication-associated protein (Rep) and showing a genomic organization typical of viruses from the family Genomoviridae. Phylogenetic analysis with deduced amino acid sequences of Rep indicates that the virus from AdO is closely related to other members of the genus Gemycircularvirus, while the virus from AdDF is related to those of the genus Gemyduguivirus. These new genomoviruses are tentatively named bemisia-associated genomovirus AdO and bemisia-associated genomovirus AdDF.

Discovery of two small circular ssDNA viruses associated with the whitefly Bemisia tabaci.

The complete genome sequences of two novel small circular DNA viruses isolated from sweet-potato whiteflies collected in central-West (AdDF) and Southeast (AdO) regions of Brazil were determined by Next Generation Sequencing (NGS), and confirmed by cloning and Sanger sequencing. The genomes are 2,199 and 2,211 nt-long, respectively, encoding a putative coat protein (CP) and a replication-associated protein (Rep) and showing a genomic organization typical of viruses from the family Genomoviridae. Phylogenetic analysis with deduced amino acid sequences of Rep indicates that the virus from AdO is closely related to other members of the genus Gemycircularvirus, while the virus from AdDF is distantly related to other genomovirus. It was thus classified in a putative new genus, for which the name "Gemybolavirus" is proposed. These new genomoviruses are tentatively named "Bemisia associated gemybolavirus AdDF", and "Bemisia associated gemycircularvirus AdO".

RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum.

The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.

Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced resistance to aphids.

Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system.

Novel biopesticide based on a spider venom peptide shows no adverse effects on honeybees.

Evidence is accumulating that commonly used pesticides are linked to decline of pollinator populations; adverse effects of three neonicotinoids on bees have led to bans on their use across the European Union. Developing insecticides that pose negligible risks to beneficial organisms such as honeybees is desirable and timely. One strategy is to use recombinant fusion proteins containing neuroactive peptides/proteins linked to a 'carrier' protein that confers oral toxicity. Hv1a/GNA (Galanthus nivalis agglutinin), containing an insect-specific spider venom calcium channel blocker (ω-hexatoxin-Hv1a) linked to snowdrop lectin (GNA) as a 'carrier', is an effective oral biopesticide towards various insect pests. Effects of Hv1a/GNA towards a non-target species, Apis mellifera, were assessed through a thorough early-tier risk assessment. Following feeding, honeybees internalized Hv1a/GNA, which reached the brain within 1 h after exposure. However, survival was only slightly affected by ingestion (LD50>100 µg bee(-1)) or injection of fusion protein. Bees fed acute (100 µg bee(-1)) or chronic (0.35 mg ml(-1)) doses of Hv1a/GNA and trained in an olfactory learning task had similar rates of learning and memory to no-pesticide controls. Larvae were unaffected, being able to degrade Hv1a/GNA. These tests suggest that Hv1a/GNA is unlikely to cause detrimental effects on honeybees, indicating that atracotoxins targeting calcium channels are potential alternatives to conventional pesticides.

Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis).

BACKGROUND: The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. In vitro directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of Bacillus thuringiensis. RESULTS: Bioassays carried out with A. grandis larvae revealed that the LC50 of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability. CONCLUSIONS: The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control A. grandis.

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut.

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a approximately 120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots.